Distinct Aggregation Mechanisms of Monoclonal Antibody Under Thermal and Freeze-Thaw Stresses Revealed by Hydrogen Exchange

Abstract

Reduced hydrogen exchange in three CDRs suggests these residues may form strong intermolecular contacts in the antibody aggregates; regions of enhanced HX indicate unfolding. Residue level modeling methods with varying levels of atomistic detail were unable to identify aggregation patterns predictively.

Rapid screening of IgG quality attributes – effects on Fc receptor binding

“…Due to the avidity effects of aggregates, the impact of a small fraction of dimers and higher oligomers in samples can alter binding to Fc receptors and can therefore not be neglected. Protein aggregates may consist of reversible and irreversible aggregates 43. Aggregates that are artificially created (heating or chemically coupling) can generally be well characterized by other analytical assays 26, 43, 44, whereas reversible aggregates of IgGs which naturally occur may fall apart upon dilution 43 and are therefore difficult to characterize.…”

“…Protein aggregates may consist of reversible and irreversible aggregates 43. Aggregates that are artificially created (heating or chemically coupling) can generally be well characterized by other analytical assays 26, 43, 44, whereas reversible aggregates of IgGs which naturally occur may fall apart upon dilution 43 and are therefore difficult to characterize. The nature of aggregates in stressed IgG samples may be different compared to naturally occurring aggregates, which complicates the assignment of the impact these have in binding assays.…”

Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody

“…Given the recent interest in the potential utility of hydrogen/deuterium exchange mass spectrometry for analysis of IgG monoclonal antibodies (mAbs) [9,[16][17][18], it is important to assess the nature and extent of peptide carry-over issues with each mAb, especially given the large number of peptides generated with these~150 kDa proteins. In this paper, we show that the primary source for carry-over for our system arose not from the reversed-phase columns, but from the online pepsin digestion process.…”

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

“…SAP analysis uses information on both the solvent-accessible area and the hydrophobicity of protein amino acid residues via molecular simulations to identify protein aggregation hot spots [33,34]. HDX and SAP analyses both pinpointed a potential aggregation interface in the C H 2 region of Fc, which further reveals the importance of the C H 2 region in the aggregation of IgG-type molecules as observed previously [28][29][30]. To the best of our knowledge, this is the first study that provides molecular-level aggregation information for a human Fc-fusion protein.…”

“…By monitoring protein amide backbone HDX, which depends on the protein local solvent accessibility and hydrogen bonds, one can characterize the solution conformation and dynamics of proteins, including changes due to the external environment [18] (e.g., chemical modifications [19][20][21][22], buffer components [23,24], and the presence of a binding protein/ligand [25,26]). Regarding the characterization of protein aggregation, HDX-MS has shown its potential for our understanding of protein self-association interfaces and aggregation-induced protein conformational changes at the molecular level in either the lyophilized state [27] or the solution state [28][29][30].…”